Process for the Preparation of Cilastatin and Sodium Salt

ABSTRACT

The present invention relates to an improved process for the preparation of Cilastatin Sodium of formula (I). The present invention also provides an isolation technique for Cilastatin acid from the reaction mixture.

FIELD OF THE INVENTION

The present invention relates to an improved process for the preparationof Cilastatin of formula (I) and its sodium salt. The present inventionalso provides a direct isolation technique for Cilastatin acid from thereaction mixture.

DESCRIPTION OF THE PRIOR ART

Cilastatin sodium is the sodium salt of a derivatized heptenoic acid.Its chemical name is[R—[R*,S*-(Z)]]-7-[(2-amino-2-carboxyethyl)thio]-2-[[(2,2-dimethylcyclopropyl)carbonyl]amino]-2-heptenoicacid, monosodium salt. It is an off-white to yellowish-white,hygroscopic, amorphous compound. PRIMAXIN (Imipenem and Cilastatin) is aformulation of Imipenem (a thienamycin antibiotic) and Cilastatinsodium.

Imipenem with Cilastatin acts as an effective antibiotic for thetreatment of infections of various body systems. PRIMAXIN is a potentbroad-spectrum antibacterial agent for intramuscular administration.Imipenem can be further described as a semi-synthetic thienamycin thatis administered intravenously or intramuscularly in combination withCilastatin to reduce toxicity. Cilastatin, a renal dipeptidaseinhibitor, inhibits the enzymatic breakdown of Imipenem and increasesurinary excretion of the active drug.

Originally Cilastatin was disclosed in U.S. Pat. No. 5,147,868. Thispatent also discloses various processes for the preparation ofCilastatin, particularly example 19A of this patent disclose a processfor the preparation of Cilastatin. According to this example thecondensation of 7-chloro-2-oxoheptanoic acid ethyl ester (I) with(S)-2,2-dimethylcyclopropanecarboxamide (II) by means of p-toluenesulphonic acid in refluxing toluene gives(S)-7-chloro-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acidethyl ester (III), which is hydrolyzed in aq. NaOH to yield thecorresponding carboxylic acid (IV). Finally, this compound is condensedwith (R)-cysteine (V) by means of NaOH in water to afford the targetCilastatin, followed by isomerisation to at 3.0 pH. The process followedin this example is depicted as below:

WO 03/018544 claims a process for the purification of Cilastatin, whichcomprises contacting a solution of crude Cilastatin with a non-ionicadsorbent resin and recovering pure Cilastatin from a solution thereof.This publication also claims a process for the isomerisation ofCilastatin by heating a solution of Cilastatin containing thecorresponding E isomer at a pH of about 0.5 to 1.5. This invention notsuitable for plant point of view as it involves column chromatography.

US 2004/0152780 claims a process for the preparation of pure Cilastatinsodium in an amorphous form which comprises recovering Cilastatin sodiumfrom a solution thereof which contains an organic solvent, homogeneousmixture of organic solvents, or homogeneous mixture of organic solventsand water, by solvent precipitation. According to this patent the pureCilastatin sodium in amorphous form was recovered from the solution ofCilastatin sodium in a solvent (where Cilastatin sodium was soluble) byadding an anti-solvent (where Cilastatin sodium was insoluble).

WO 2006/022511 claims a process for preparing Cilastatin sodium viaCilastatin amine salt, also the said patent claims Cilastatin ammoniumsalt. However EP 0 048 301 page 2; line 33-37 & U.S. Pat. No. 4,616,038col 36; 40-44 anticipates the claim of the said publication. Also thispatent utilizes the column chromatography for removing sodium chloride.

However taking the consideration the commercial importance of Cilastatinsodium and Imipenem, there remains a need of convenient process. Hence,we focused our research to find an alternative processes and succeededwith a process that eliminates the foregoing problems associated withearlier processes.

OBJECTIVE OF THE INVENTION

The primary objective of the present invention is to provide acommercial process for the preparation of Cilastatin.

Another objective of the present invention is to provide a process forthe preparation Cilastatin sodium with high purity and in good yields.

Still another objective is to provide a process which obviates the useof chromatography and provides direct isolation of Cilastatin.

Yet another objective of the present invention provides a process forthe isomerisation of7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoicacid.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides an improved process forpreparation of Cilastatin or its salt of formula (I)

which comprises the steps of:

-   -   (i) condensing        7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic        acid of general formula (II) with L-Cysteine in the presence of        base in water or an alcoholic solvent or aqueous alcoholic        solvent, and    -   (ii) acidifying and isolating the Cilastatin acid (I).

wherein X represents a leaving group selected from chloro or bromo.

Another embodiment of the present invention provides a process for thepreparation of Cilastatin Sodium

-   -   a) dissolving Cilastatin acid in a solvent using an organic        base,    -   b) adding sodium salt of weak acid, and    -   c) isolating Cilastatin Sodium.

The present invention further provides an improved process for thepreparation of pure Cilastatin Acid of formula (I), which comprises thesteps of:

-   -   i) condensing        7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic        acid of formula (I) with L-Cysteine in the presence of base an        alcoholic solvent or aqueous alcoholic solvent,    -   ii) optionally removing NaCl or KCl by filtration and        concentrating filtrate,    -   iii) optionally adding water,    -   iv) adjusting pH to 2.0 to 4.0,    -   v) optionally extracting the Cilastatin acid into C₄-C₈ alcohol        selected form group comprising of n-butanol, cyclohexanol, and    -   vi) isolating Cilastatin acid.

DETAILED DESCRIPTION OF THE INVENTION

In an embodiment of the present invention, the base used in step (i) isselected from sodium hydroxide, potassium hydroxide, sodium carbonateand the like, and alcoholic solvent used in step (i) is selected frommethanol, ethanol, isopropanol and the like. It has been found that thecondensation of compound of formula (II) with L-Cysteine in thealcoholic solvent medium yields Cilastatin in pure form, wherein theimpurity formation is negligible.

In still another embodiment of the present invention, the L-Cysteineused in step (i) is in the form of L-Cysteine hydrochloride monohydrateor R-Cysteine hydrochloride or L-Cysteine hydrobromide monohydrate.

In another embodiment of the present invention, the pH of reaction massis adjusted to 2.0 to 4.0 using hydrochloric acid, sulfuric acid, formicacid, acetic acid, trifluoracetic acid and the like.

In another embodiment of the present invention, there is provided aprocess for isolation/crystallization of Cilastatin acid withoututilizing the column chromatography, which crystallizing Cilastatin froma solution thereof which contains an water miscible organic solvent,water, or a mixture thereof.

According to this invention the aqueous solution of Cilastatincontaining impurities which may be inorganic salts such as sodiumchloride, sodium bromide, sodium formate, sodium acetate and the like,or organic impurities which may have formed due to the degradation ofCilastatin, or the side products formed during the synthesis, orunreacted intermediates is stirred at a pH in the range of 4.0 to 2.0for a long period, the solid crystallized was filtered and if requiredsubjected to purification.

In another aspect the inorganic salts like NaCl, KCl is filtered formthe reaction mass, if the condensation taken place in an alcoholicsolvent medium, prior to pH adjustment. The solution of crude Cilastatincan be directly taken from the reaction mass or may be obtained bydissolving crude Cilastatin in water and water miscible solvent. It hasbeen noted that the conventional methods requires column chromatographyto remove the inorganic salt like sodium chloride that are formed duringthe course of reaction, whereas the present invention provides a processin which Cilastatin is directly crystallized from the reaction mass atthe pH in the range of 2.0 to 4.0 more particularly at the pH in therange of 3.0.

In one more embodiment of the present invention the Cilastatin can beprecipitated from reaction mass after pH adjustment by stirring thesolution for a long period or it can be extracted into solvents likeC₄-C₈ alcohol selected form group comprising of n-butanol, cyclohexanol,followed by isolating Cilastatin acid form the resultant layer. Inanother aspect the Cilastatin acid is isolated from the C₄-C₈ alcohollayer by reducing the volume of the layer followed by filtering theprecipitated Cilastatin acid, or by evaporating the layer followed bycrystallizing Cilastatin acid from mixture of water and water-misciblesolvent.

In yet another embodiment of the present invention, water-misciblesolvent used solvent used during crystallizing Cilastatin sodium areselected from acetone, methanol, THF, n-butanol, acetonitrile, DMF, andit can be vary from 5% to 95%. In another embodiment of the presentinvention the Cilastatin obtained may contain up to 10% NaCl or about0.5% NaCl, preferably 2% to 0.5% of NaCl.

In still another embodiment of the present invention, the Cilastatinobtained according to the present invention can be optionally subjectedto isomerisation process by the technique known in prior art.

In still another embodiment of the present invention, the startingmaterial7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic acidof formula (II) is prepared by utilizing the process available in theprior art.

In one more embodiment of the present invention, the present inventionprovides a process for isomerisation of E-isomer of7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoicacid, Cilastatin precursor which comprises treating the mixture of E andZ-7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoicacid in a solvent selected from toluene, MDC, water and the like ormixtures thereof with HCl at a pH in the range of 0.5 to 4.5. The saidisomerisation can be done at a temperature in the range of 10° C. toreflux temperature of the solvent, preferably room temperature i.e. 27to 35° C.

In yet another embodiment of this present invention, the solvent usedfor the dissolution of Cilastatin acid is selected from ethanol,isopropanol, n-butanol, denatured sprit, acetone, THF, acetonitrile, DMFand the like or mixtures thereof. Thus present invention provides anovel process for the preparation of Cilastatin sodium. The solventsystem is chosen such a way that the Cilastatin Sodium per se isinsoluble in the solvent system

In yet another embodiment of this present invention, the base used instep (a) is selected from DBU, DBN, DABCO, TMG, triethyl amine (TEA),DEA, diisopropyl amine, NaOH and the like.

In one more embodiment of the present invention the step (a) solution issubjected to carbon treatment (optional) and micron filtration to getthe Cilastatin sodium as sterile product. Accordingly this presentinvention provides a process for the preparation of Sterile CilastatinSodium.

In still another embodiment of the present invention, the salt formingagent in step (b) is selected from sodium lactate, sodium acetate,sodium 2-ethyl hexanoate, and the like or the mixture thereof. Inanother embodiment of the present invention the salt forming agent canbe added directly or by dissolving the salt forming agent using step (a)solvent.

The present invention is exemplified by the following examples, whichare provided for illustration only and should not be construed to limitthe scope of the invention.

EXAMPLE 1 Preparation of 7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic Acid (II) (Starting Material)

To the solution of S-2,2-dimethylcylopropyl carboxamide (100 gm) intoluene (500) was added Ethyl-7-chloro-2-oxo-heptanoate (270 gm) andp-toluene sulphonic acid (1.5 gm). The resulted solution was refluxedfor 20 hrs azeotropically. The resulted mass was cooled to 5-10° C. andadded the solution of sodium hydroxide (140 gm) in water 500 ml and theresulted two-layered solution was stirred for 8 hrs at 25-30° C. up tothe complete disappearance of ester. The toluene layer was separated andthe aqueous layer was washed with toluene. The pH of the aqueous layerwas adjusted to 4.0 to 4.5 and extracted with toluene (1 lt). Thetoluene layer containing 7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic acid was washed with water andused as such for the next step. The ratio of Z and E isomer 90:10% wasobtained.

EXAMPLE 2 Isomerisation of 7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic Acid (II)

To the toluene layer, obtained from example −1, was added hydrochloricacid (1 It) and stirred for 4 hrs at 25-30° C. till the disappearance ofE isomer. The toluene layer was separated and washed with water andfollowed by brine. The toluene layer was distilled out under vacuum upto 50% of the original volume. To the reaction mass hexane/IPE was addedat 50° C. and cooled to 0-5° C. The precipitated mass was filtered andwashed with hexane (200 ml) and dried under vacuum to obtained 99% pureZ-7-chloro-2[[(1S)-2,2-dimethyl cyclopropane]carboxamide]-2-heptenoicacid (150 gm) as white solid.

EXAMPLE 3 Preparation of Cilastatin Acid (I)

To the solution of sodium hydroxide (90 gm) in water (1 lt) was addedL-Cysteine hydrochloride monohydrate (96 gm) andZ-7-chloro-2[[(1S)-2,2-dimethyl cyclopropane]carboxamide]-2-heptenoicacid and stirred at 25-30° C. till the disappearance ofZ-7-chloro-2[[(1S)-2,2-dimethyl cyclopropane]carboxamide]-2-heptenoicacid. After completion of reaction, the reaction mass was washed withdichloromethane (500 ml. To the aqueous layer was added carbon (10 gm)and stirred and filtered. To the filtrate was added water (1 lt) and thepH of the solution was adjusted to 3.0 and stirred for 24 hrs. Theprecipitated mass was filtered, washed with water (200 ml) and withacetone (500 ml) and dried to obtain 10 gm white solid with 97% purity.The solid was dissolved in water (700 ml) and added MDC (700 ml) andethyl acetate (100 ml) and stirred for 10 hrs. The precipitated mass wasfiltered and washed with water (100 ml) and acetone (200 ml) and driedto obtain 100 gm white Cilastatin acid with 99.5% purity.

EXAMPLE 4 Preparation of Cilastatin Sodium

The Cilastatin acid (100 gm, 99.5%) was dissolved in the mixture ofethanol (2.5 lt) and triethylamine (30 gm) at 25 to 30° C. To theresulted clear solution was added carbon (10 gm) and stirred andfiltered. The filtrated was filtered again through sterile micron (0.2μ)filter. To the resulted clear solution was added solution of sodiumethyl hexanoate (70 gm) in ethanol (70 ml) and stirred for 3 hrs at 25to 30° C. The precipitated Cilastatin sodium was filtered and washedwith ethanol (80 ml) and followed by acetone (200 ml) and dried undervacuum to obtained 95 gm Cilastatin sodium as amorphous white solid with99.5% purity.

EXAMPLE 5 Preparation of Cilastatin Acid

To the solution of sodium hydroxide (88 gm) in methanol (1500 ml) wasadded Z-7-chloro-2[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic acid and stirred to dissolve. Tothe resulted clear solution was added L-Cysteine hydrochloridemonohydrate (97 gm) and stirred the resulted suspension at 60 to 65° C.till the disappearance of Z-7-chloro-2[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic acid. After completion ofreaction, the pH insoluble salts were filtered. The filtrate wasdistilled out under vacuum. The residue was dissolved in water (500 ml)and washed with dichloromethane (500 ml). The pH of aqueous layer wasadjusted to 3 to 4 from the original pH in the range of 5.5, and withn-butanol (500 ml). The butanol layer was washed with water anddistilled. The residue was dissolved in water (100 ml) and addedacetonitrile (1500 ml) at 50° C. and further refluxed at 80° C. for onehr. The precipitated cilastatin acid was filtered and washed withacetonitrile (100 ml). The crude wet cake (60 gm) was refluxed withacetonitrile water mixture (9:1,1500 ml), and cooled to yield 60 gm purecilastatin acid with 99.5% purity.

EXAMPLE 6 Preparation of Cilastatin Acid

To the solution of sodium hydroxide (88 gm) in methanol (1500 ml) wasadded Z-7-chloro-2[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic acid and stirred to dissolve. Tothe resulted clear solution was added L-Cysteine hydrochloridemonohydrate (97 gm) and stirred the resulted suspension at 60 to 65° C.till the disappearance of Z-7-chloro-2[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic acid. The pH of the reaction masswas adjusted to 7.0 with conc.HCl and filtered the insoluble salts. Thefiltrated was distilled out under vacuum. The residue was dissolved inwater (500 ml) and washed with dichloromethane (500 ml). The pH ofaqueous layer was adjusted to 3 to 4 from the original pH in the rangeof 5.5, and with n-butanol (500 ml). The butanol layer was washed withwater and distilled up to 50% of original volume and stirred at 25° C.The precipitated cilastatin acid was filtered and washed with n-butanol(100 ml) followed by acetone to yield 60 gm pure cilastatin acid with99.7% purity.

EXAMPLE 7 Preparation of Cilastatin Sodium

The Cilastatin acid (100 gm, 99.5%) was dissolved in the mixture ofn-butanol (2.5 lt) and triethylamine (30 gm) at 25 to 30° C. To theresulted clear solution was added carbon (10 gm) and stirred andfiltered. The filtrated was filtered again through sterile micron (0.2μ)filter. To the resulted clear solution was added solution of sodiumethyl hexanoate (70 gm) in n-butanol (70 ml) and stirred for 3 hrs at 25to 30° C. The precipitated Cilastatin sodium was filtered and washedwith n-butanol (80 ml) and followed by acetone (200 ml) and dried undervacuum to obtained 80 gm Cilastatin sodium as amorphous white solid with99.78% purity.

Abbreviations;

DBU: diazabicyclo[5,4,0]undec-7-enDBN: 1,5-diazabicyclo[4,3,0]-non-5-eneTMG: 1,1,3,3-tetramethylguanidineDABCO: 1,4-diazabicyclo-[2,2,2]-octane

1) An improved process for the preparation of Cilastatin Sodium of formula (I), which comprises the steps of: (i) condensing 7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic acid of general formula (II)

wherein X represents a leaving group selected from chloro or bromo; with L-Cysteine derivative of formula

in the presence of base in water or an alcoholic solvent or aqueous alcoholic solvent, (ii) acidifying and isolating the Cilastatin acid by filtration, (iii) dissolving Cilastatin acid in a solvent using an organic base, (iv) adding sodium salt of weak acid, and (v) isolating Cilastatin Sodium. 2) An improved process for the preparation of pure Cilastatin Acid of formula (I), which comprises the steps of: i) condensing 7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic acid of formula (I) with L-Cysteine in the presence of base and an alcoholic solvent or aqueous alcoholic solvent, ii) optionally removing inorganic salt by filtration and concentrating filtrate, iii) optionally adding water, iv) adjusting pH to 2.0 to 4.0, v) optionally extracting the Cilastatin acid into C4-C8 alcohol selected form group comprising of n-butanol, cyclohexanol, and vi) isolating Cilastatin acid. 3) A process as claimed in claim 1, wherein the alcoholic solvent used in step (i) is selected from methanol, ethanol, iso-propanol, or n-butanol; and base used in step (i) is selected from sodium hydroxide, potassium hydroxide, or sodium carbonate. 4) A process as claimed in claim 1, wherein the isolation of Cilastatin acid is carried out by, stirring the resultant solution obtained after acidification or pH adjustment for a long period, followed by filtering the solid. 5) A process as claimed in claim 2, wherein the isolation of Cilastatin acid is carried out by reducing the C₄-C₈ alcohol to half of the original volume, followed by filtration. 6) A process as claimed in claim 2, wherein the isolation of Cilastatin acid is carried out by concentrating the C₄-C₈ alcohol layer, followed by crystallizing the solid. 7) A process for direct isolation of Cilastatin acid by crystallizing Cilastatin acid at a pH in the range of 2.0 to 4.0 from a reaction mass containing water-miscible organic solvent, water, or a mixture thereof and/or sodium chloride or sodium bromide and/or other inorganic or organic impurities. 8) A process as claimed in claim 6, wherein the water-miscible organic solvent is selected from methanol, ethanol, isopropanol, n-butanol, acetone, THF, acetonitrile, or DMF. 9) An improved process for the preparation of Cilastatin Sodium which comprising the steps of: a) dissolving Cilastatin acid in a solvent using an organic base, b) adding sodium salt of weak acid, and c) isolating Cilastatin Sodium. 10) The process as claimed in claim 1, wherein the organic base used in step (iv or a) is selected from DBU, DBN, TMG, trimethyl amine (TEA), DEA, or diisopropyl amine; and the solvent used is selected from ethanol, n-butanol, denatured sprit, acetone, THF, acetonitrile or mixtures thereof. 11) The process as claimed in claim 1, wherein the source of sodium salt of weak acid used in step (iv or b) is selected from sodium lactate, sodium acetate, sodium 2-ethyl hexanoate, sodium acetate or mixtures thereof. 12) A process for the isomerisation of 7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic acid, the said process treating the 7-chloro-2-[[(1S)-2,2-dimethylcyclopropane]carboxamide]-2-heptenoic acid in a solvent selected from a group comprising of toluene, MDC, water and the like or mixtures thereof with dil HCl at a pH in the range of 0.5 to 2.0. 13) A process as claimed in claim 2, wherein the alcoholic solvent used in step (i) is selected from methanol, ethanol, iso-propanol, or n-butanol; and base used in step (i) is selected from sodium hydroxide, potassium hydroxide, or sodium carbonate. 14) A process as claimed in claim 2, wherein the isolation of Cilastatin acid is carried out by, stirring the resultant solution obtained after acidification or pH adjustment for a long period, followed by filtering the solid. 15) The process as claimed in claim 9, wherein the organic base used in step (iv or a) is selected from DBU, DBN, TMG, trimethyl amine (TEA), DEA, or diisopropyl amine; and the solvent used is selected from ethanol, n-butanol, denatured sprit, acetone, THF, acetonitrile or mixtures thereof. 16) The process as claimed in claim 9, wherein the source of sodium salt of weak acid used in step (iv or b) is selected from sodium lactate, sodium acetate, sodium 2-ethyl hexanoate, sodium acetate or mixtures thereof. 